Thermal surgical procedures and compositions

ABSTRACT

Methods, compositions, and systems useful to enhance a thermal surgical procedure are described. Compositions include at least one compound effective to induce an inflammatory response in biological material identified to undergo a thermal surgical procedure. Methods and systems include providing compositions of the invention to biological materials and treating biological materials with an inflammation inducing composition for a time, amount, and type effective to induce inflammation in at least a portion of the biological material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/457,691, filed Mar. 26, 2003, the entire content of which isincorporated herein by reference.

GOVERNMENT FUNDING

The present invention was made with support from National Institutes ofHealth under Grant No. R29CA75284. The U.S. government may have certainrights in this invention.

TECHNICAL FIELD

The present invention relates generally to thermal surgical procedures.

BACKGROUND OF THE INVENTION

Thermal surgical procedures, wherein thermal energy is either withdrawnfrom and/or delivered to a localized region of biological material in aneffort to destroy the region of biological material, are known in theart and have been shown to be an effective treatment of disease,particularly in instances wherein a patient is unwilling or unable toundergo another form of surgery. A thermal surgical procedure mayinclude, for example, a cryosurgical procedure in which thermal energyis removed from biological material to cool and/or freeze the biologicalmaterial in an effort to destroy it. Such procedures have been routinelyused to treat malignancies on the surface of the body and is also usedfor treating and managing malignancies of internal organs, such askidney and prostate. Also, a thermal surgical procedure can include aprocedure in which thermal energy is added to biological material toheat the biological material in an effort to destroy it. The destructionof biological material may or may not result in ablation of some or allof the biological material. Thermal surgical procedures are useful intreating diseases of various tissues, including, for example, carcinomasof the liver, kidney, and prostate. These techniques are advantageous inthat they have the potential for less invasiveness and lower morbidityas compared with surgical excision.

Thermal surgical procedures involving delivery of thermal energy toincrease the local temperature of biological material above thephysiological temperature is known to be an effective treatment foreliminating malignant tissue. Typical temperatures for thermal surgicalprocedures involving the delivery of thermal energy are at or above 50degrees Celsius (° C). Typically, the biological material is heated toelevated temperatures and is maintained at these temperatures for aninterval of several minutes.

Additionally, it is known in the art that freezing biological materialsis an effective method for controlling and destroying the cells andtissues of, for example carcinomas of various tissues and organs.Cryosurgical techniques, in combination with monitoring techniques, suchas ultrasound and MRI, have provided effective treatment of a number ofinternal organs, including liver, prostate, and kidneys. Results ofcryosurgery involving carcinomas in kidneys suggest that this may provea useful technique, particularly for small renal cell carcinomas.Cryosurgical procedures typically reduce the temperature of thebiological material to temperatures close to or below the temperature atwhich the biological material will freeze, often below 0° C. and as lowas −20 to −60° C. Typically the biological material is cooled to andmaintained at these temperatures for an interval of minutes.

Nonetheless, there exists clinical evidence of recurrence of disease inthermal surgically treated patients. This result may be due to theinitial challenge of treating the entire diseased tissue. For example,in current thermal surgical procedures it is prudent to take asufficient surgical margin around diseased tissue to ensure all of themalignant tissue is removed or destroyed. This often involves freezingor heating beyond a tumor and invading surrounding normal tissue.However, care must be taken to not invade too far beyond the diseasedtissues, particularly when treating biological material near healthysensitive tissues. In particular, when treating prostate cancers, whichoccur principally in the peripheral zone of the prostate near a numberof sensitive structures, such as the rectum, bladder, externalsphincter, and the cavernosal nerves, a surgeon must be careful to sparethe surrounding tissues from injury. This is particularly important intreating the prostate where overfreezing into the areas of the rectumand urethra can cause rectal and urethral fistulas. On the other hand,if a surgeon is too conservative and underheats or underfreezes affectedtissues, the disease may not be effectively treated and the likelihoodof recurrence of the disease increases.

There is a need in the art, therefore, to improve the clinicalapplication of thermal surgical procedures, including effectivelymonitoring of the heating or freezing of the biological material to moreeffectively predict the zone of injury, reproducibly creating andenhancing cell death within the heat treated area or the cryolesion, andimproving definition of the edge of the heat treated area or thecryolesion to improve the effectiveness of the kill zone whileprotecting adjacent normal tissues.

SUMMARY OF THE INVENTION

The present invention provides a composition, method, system, and/or kitfor use in a thermal surgical procedure. As used herein thermal surgicalprocedures generally include, but are not limited to, surgicalprocedures in which thermal energy is either withdrawn from and/ordelivered to a localized region of biological material in an effort todestroy at least a portion of biological material.

The compositions of the present invention include one or more compoundsthat can effectively induce an inflammation response in biologicalmaterials involved in the thermal surgical procedure. A “compound” asused herein, may include a single constituent or a combination of two ormore constituents. Furthermore, a “composition” as used herein mayinclude only one compound or combination of two or more compounds.

Biological materials that may be treated using the compositions,methods, and systems of the present invention include, but are notlimited to, cells, tumor cells, tissue, tumor tissues, tissues ofinternal organs such as liver tissue, prostate tissues, breast tissue,and kidney tissues. In addition, biological materials may also include,but are not limited to vascular tissues, gastrointestinal tissues,muscle tissues, including myocardium, tissues of the skin, andconnective tissues. Combinations of these biological materials in situare possible, and treatment of some biological materials to theexclusion of others is also contemplated.

The present invention may be used in the treatment of various cancersand/or tumors such as, but not limited to, prostate cancer, livercancer, kidney cancer, breast cancer, uterine fibroids, as well as anyother tumor or tissue where thermal surgical procedures have typicallybeen used or which may be found useful in the future. The presentinvention may also be useful in the treatment of benign prostatichypertrophy (BPH), or treatment of stenosis of the urethra. In addition,the present invention may also be useful in treating any number ofautoimmune and chronic inflammatory disorders, where the associatedtissues involved in the disorder are predisposed to injury from coolingor heating. Examples include, but are not limited to, rheumatoidarthritic syndrome, emphysema, pulmonary hypertension and cardiacfailure, Crohn's disease, neurological disorders that displayneuroinflammatory disease, ulcerative colitis, and other knownautoimmune diseases.

In addition, the present invention may also be useful in any number ofinterventional procedures that are currently used to treat individuals.For example, the present invention may be useful in procedures thatutilize cooling or heating to destroy biological materials. Thus, thepresent invention may be used in conjunction with thermal surgicalprocedures performed on myocardial tissue for treating rhythmirregularities of the heart. Further, the present invention may be usedin preventing restenosis of arteries treated with angioplasty,atherectomy, or other procedures for opening occlusions in thevasculature.

In one aspect, the present invention includes a method of performing athermal surgical procedure, wherein the method includes: identifyingbiological material to undergo the thermal surgical procedure;contacting the biological material with an inflammation inducingcomposition, wherein inflammation is induced in at least a portion ofthe identified biological material; and adjusting the temperature of theidentified biological material, wherein at least a portion of thebiological material is destroyed after undergoing the thermal surgicalprocedure. The temperature may be adjusted above a physiologicaltemperature of the biological material, a thermosurgical procedure, orthe temperature may be adjusted to below a physiological temperature, asin a cryosurgical procedure. It is contemplated that a thermal surgicalprocedure may also include both a thermosurgical procedure and acryosurgical procedure, either on the same identified biologicalmaterial with the procedures performed at separate times, or on separatesites of the identified biological material.

In another aspect, the present invention includes a composition thatincludes at least one compound effective for inducing an inflammatoryresponse in biological material that is identified to undergo a thermalsurgical procedure. The composition may include a single constituent asthe active ingredient, or may include a combination of activeingredients. Furthermore, the composition may also include such optionalconstituents as a physiological carrier and/or a buffering agent.

In a further aspect, the present invention provides a method ofperforming a thermal surgical procedure for biological material, whereinthe thermal surgical procedure may be a thermosurgical procedure, acryosurgical procedure, or any combination thereof. The thermal surgicalprocedure includes: identifying biological material to be treated priorto a thermal surgical procedure; contacting the biological material withan inflammation inducing composition for a time, amount and typeeffective to induce inflammation in at least a portion of the biologicalmaterial, wherein inflammation is induced in at least a portion of theidentified biological material; and adjusting the temperature of theidentified biological material, wherein at least a portion of thebiological material is destroyed after undergoing the thermal surgicalprocedure.

The present invention additionally provides a system for inducinginflammation in biological material identified to undergo a thermalsurgical procedure. This system generally includes: a compositionincluding at least one compound effective for inducing inflammation inat least a portion of the biological material; and means for deliveringthe composition to a least a portion of the biological material. Thecomposition may include a single active ingredient or more than oneactive ingredient, and may further include optional constituents, suchas a pharmaceutically acceptable carrier and/or a buffering agent.

In an additional aspect, the present invention provides methods oftreating diseases, such as cancer. A method of treating cancer isdisclosed which includes: identifying a localized region of a mammalcomprising biological material further including cancer; providing to atleast a portion of the biological material a composition comprising asan active ingredient at least one compound for a time, amount and typeeffective to induce inflammation in at least a portion of the biologicalmaterial, thereby providing inflamed biological material; and applying athermal surgical procedure to at least a portion of the inflamedbiological material. The thermal surgical procedure may be athermosurgical procedure, a cryosurgical procedure, or a combinationthereof.

Additional diseases may be treated by the methods and compositions ofthe present invention. For example, the present invention includes amethod of treating a disease that includes: identifying a localizedregion of a mammal comprising biological material typical of thedisease; providing to at least a portion of the biological material acomposition comprising as an active ingredient at least one compound fora time, amount and type effective to induce inflammation in at least aportion of the biological material, thereby providing inflamedbiological material; and

applying a thermal surgical procedure to at least a portion of theinflamed biological material.

The present invention further discloses a kit for use in a thermalsurgical procedure. Such kit includes, generally: a thermal surgicalprobe adapted to transfer thermal energy; and a composition comprisingat least one compound effective for inducing an inflammatory response inbiological material identified to undergo a thermal surgical procedure.

The above summary of the present invention is not intended to describeeach embodiment or every implementation of the present invention.Advantages, together with a more complete understanding of theinvention, may become apparent and appreciated by referring to thefollowing detailed description of illustrative embodiments taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows one embodiment of a system according to the presentinvention.

FIG. 2 shows one example of a relationship of temperature versusdistance from the center of an ice ball according to the presentinvention.

FIG. 3 shows a cross-sectional view of one example of an ice ballaccording to the present invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the following detailed description of certain illustrativeembodiments, reference is made to drawings that form a part hereof, andin which are shown by way of illustration, certain embodiments throughwhich the invention may be practiced. It is to be understood that otherembodiments may be utilized and processing steps/structural changes maybe made without departing from the scope of the present invention.

As will be discussed below, the present invention provides methods,compositions, and systems for use in treating biological materialsincluding, for example, cells, tissues, and combinations thereof, with athermal surgical procedure. The present invention generally includes aninflammation inducing composition that can be used to treat thebiological material that is the subject of the thermal surgicalprocedure, wherein the biological material to undergo a thermal surgicalprocedure may be identified, in whole or a portion thereof, and istreated with an inflammation inducing composition of the presentinvention for a time, in an amount, and of a type effective to induce atleast some inflammation in at least a portion of the identifiedbiological material. The present invention further includes methods andsystems for inducing such inflammation. The inflammation inducingcomposition of the present invention can be used to induce aninflammatory response in the biological material, where the inducedinflammatory response may provide for an enhancement of destruction ofthe biological material during the thermal surgical procedure.

The methods and compositions of the present invention may be useful intreating several diseases, including cancer. Cancers that maypotentially be treated by the present methods and compositions include,but are not limited to, cancers of internal organs, such as prostate,liver, and kidney, cancers of bone and cartilage, skin cancer, oralcancer, musculoskeletal cancers, breast cancer, gynecological cancersincluding uterine fibroids. Other diseases that may benefit from themethods and compositions of the present invention include, for example,benign prostatic hypertrophy, stenosis of the urethra, rheumatoidarthritic syndrome, emphysema, pulmonary hypertension, cardiac failure,Crohn's disease, neurological disorders displaying neuroinflammatorydisease, ulcerative colitis, and gynecological disorders.

Inflammation is understood herein to typically involve a complex seriesof events which may include, but is not limited to, dilatation ofarterioles, capillaries and venules, with increased vessel permeabilityand blood flow, exudation of fluids through the vessel walls, includingplasma proteins and leucocytic migration into the inflammatory focus.Further, on a molecular level adhesion molecules are typicallyupregulated in endothelial cells which slow down (by rolling) andcapture (by adhesion) leucocytes to the vessel walls in the area ofinflammation.

Without wishing to be bound by any particular theory, it is believedthat an induced inflammatory response in biological materials intendedto undergo a thermal surgical procedure may augment the effectiveness ofthat procedure. It is further believed that by controlling the extentand degree of the induced inflammatory response in the biologicalmaterial, the degree to which the thermal surgical procedure will besuccessful may be significantly influenced. Using compounds that, forexample, induce non-destructive inflammation in biological materialprovides beneficial changes in the effectiveness of thermal surgicalprocedures as compared to untreated biological material. Thus, thepresent invention provides improvements in typical thermal surgicalprocedures by providing what is believed to be a controllable andreproducible technique to accentuate the injury and death of biologicalmaterial undergoing the thermal surgical procedure.

Thermal surgical procedures have been shown to be effective treatmentmodalities for several tumor tissues. For example, cryosurgicalprocedures are known to be effective treatments for eliminatingmalignant tissue. In cryosurgical procedures, thermal energy is removedfrom at least a portion of the biological material so as to decrease thelocal temperature below a physiological temperature of the biologicalmaterial. A physiological temperature of the biological material isgenerally understood to be that temperature at which the physicalmechanisms of living organisms and/or parts thereof are able tofunction. Cryosurgical procedures reduce the temperature of thebiological material to temperatures close to and/or below thetemperature at which the biological material will freeze. Typicaltemperatures for cryosurgical procedures include those at or below 0°C., and may further include temperatures at or below approximately −20°C., going down to at or below −60° C. The biological material may becooled to and maintained at these temperatures for, e.g., an interval ofminutes, or any other suitable period of time, to ensure effectivenessof the treatment.

While in a cryosurgical procedure it may be preferred to take asufficient surgical margin around the malignant tissue to ensure thatall tumor tissue has been removed or destroyed, often requiring freezingbeyond the tumor into normal tissue, the present invention is believedto reduce the potential side effects of normal tissue damage duringcryosurgery, and to maximize the tumor destruction at the edge of thecryosurgical ice ball, strategies to both protect (e.g., normal) andsensitize (e.g., tumor) cells to freezing. Protecting and/or sensitizingtissues from temperatures experienced within the ice ball may allowsurgeons to functionally increase the surgical margin while decreasingdamage to surrounding tissues. Also, increasing the efficiency of tissuedestruction within the ice ball may increase the confidence that anincreased number of, e.g., tumor cells are killed near the periphery ofthe tissue of interest while decreasing the chances of over-freezedamage into adjacent normal tissues, such as, e.g., the rectum, inprostate cryosurgery.

It is also believed that the present invention may provide for betterassessment of the actual location of cell and tissue death in the iceball formed during a cryosurgical procedure. Assessment of this locationcan be based in part on the region of biological material undergoing aninflammatory response induced by the use of the inflammation inducingcomposition of the present invention. Use of the inflammation inducingcomposition of the present invention may allow for a greater percentageof cell and/or tissue destruction during the cryosurgical procedure.

Thermal surgical procedures that deliver thermal energy to biologicalmaterial, understood herein as “thermosurgical procedures” are alsoknown to be effective treatments for eliminating malignant tissue. Inthese thermosurgical procedures, thermal energy is supplied to at leasta portion the biological material so as to increase the localtemperature above a physiological temperature of the biologicalmaterial. For example, typical temperatures for these thermosurgicalprocedures generally include those at or above 50° C. It is noted thatby the methods and composition of the present invention, it may bepossible to perform effective thermosurgical procedures at temperaturesbelow 50° C., such as temperatures no greater than about 40° C., therebypreventing injury to adjacent tissues. The biological material may beheated to and maintained at these temperatures for, e.g., an interval ofminutes, or any other suitable period of time, to ensure effectivenessof the treatment.

As with cryosurgery, it is typically considered to be beneficial to takea sufficient surgical margin around the biological material of interestthrough the use of the heat to ensure that all the biological materialof interest has been removed or destroyed. However, taking a sufficientmargin around the biological material typically requires heating beyondthe biological material of interest into normal tissue. To minimize thepotential side effects of normal tissue damage during a heat-deliveringthermal surgical procedure, and to maximize the destruction at the edgeof the heated biological tissue, strategies to both protect (e.g.,normal) and sensitize (e.g., tumor) cells to heating are also desirable.

The present invention is believed to sensitize tissues to and/or protecttissues from temperatures experienced at either the edge of the ice ballor the edge of the heated biological material. As a result, surgeonsperforming thermal surgical procedures according to the presentinvention could potentially functionally increase the surgical marginwhile decreasing damage to surrounding tissues by increasing theefficiency of tissue destruction at the edge of the ice ball or the edgeof the heated biological tissue and may also obtain better assessment ofthe actual location of cell and tissue death in the ice ball formedduring a cryosurgical procedures and/or the actual location of cell andtissue death at the edge of the heated biological tissue formed duringthe thermal surgical procedure.

Improvement of the assessment of the actual location of cell and/ortissue death, according to the present invention, is believed to bebased in part on the region of biological material undergoing aninflammatory response induced by the use of the inflammation inducingcomposition of the present invention. Use of the inflammation inducingcomposition of the present invention is believed to provide a greaterpercentage of cell and/or tissue destruction during the thermal surgicalprocedures contemplated by the present invention.

Generally, the compositions of the present invention may be used in alocalized region of a mammal involved in the thermal surgical procedure.Typically, the composition includes as an active ingredient at least onecompound effective to induce at least some inflammation in at least aportion of the biological material of interest, such as any native orartificial tissue of a mammal, where the at least one compound can beeffective to induce inflammation in at least a portion of the localizedregion of the native or artificial tissue of the mammal.

A composition of the present invention typically induces anon-destructive inflammation within the biological material of interest,either localized to the entire region of the thermal surgical site orlocalized to one or more portions of the thermal surgical site, priorto, during, and/or after the thermal surgical procedure. For example, acomposition of the present invention may be used to induce aninflammatory response in a localized region of the biological materialprior to or concurrent with a thermal surgical procedure. Furthermore, acomposition of the present invention may be used in a localized regionof a mammal to treat biological material, for a time, and in an amountand using a type of composition effective to induce inflammation in thematerial, that has previously been identified and has undergone athermal surgical procedure.

While not wishing to be bound by a particular theory, it is believedthat the composition of the present invention may induce thisnon-destructive inflammation by altering the behavior of vascularendothelial cells present in biological material. In particular, it isbelieved that the inflammation induced by the compositions of thepresent invention may injure the microvasculature of the biologicaltissue prior to the thermal surgical procedure. It is also believed thatthis induced inflammatory injury may precondition the microvasculatureso that it “shuts down” after the thermal surgical procedure. Incombination with the thermal surgical procedure, it is believed that theuse of the composition of the present invention may provide a moreeffective destruction of the biological material undergoing a thermalsurgical procedure by enhancing the effects of the procedure than wouldbe provided by the thermal surgical procedure performed alone withoutbenefit of inflaming the biological material.

Without being held to any particular theory, it is believed that throughan understanding of the nature of cell injury, it may be possible toaccentuate the mechanisms of injury utilizing targeted molecularadjuvants such as those described herein. It is further believed thattwo biophysical changes that occur in cells during freezing, osmoticdehydration of cells and intracellular ice formation (IIF) may be linkedto cell injury. At low cooling rates, as the freezing propagatesextracellularly, the solute concentration outside the cell begins torise, causing osmotic dehydration of the cells. As the solutes becomeconcentrated within the cells, the high concentration of solute has beenhypothesized to injure the cell in several ways including damage to theenzymatic machinery and destabilization of the cell membrane.

The second biophysical response, IIF, is believed to occur when thecooling rate is sufficiently rapid to trap water within the cell. Inthis case, the cell cannot osmotically equilibrate with theextracellular space. As a result, the cytoplasm cools and ice ultimatelynucleates within the cell, the ice crystals causing injury to theorganelles and membranes.

Damage due to solute effects is believed to typically happen atrelatively low cooling rates when the cells have sufficient time todehydrate substantially completely. IF damage, on the other hand, isbelieved to typically occur at relatively high cooling rates, when thewater is trapped inside the cells. This results in an “inverse U curve”of cell viability with low viability at extremely high and low coolingrates, and high viability at cooling rates between the extremes. Thiscooling rate behavior is highly cell-type dependent with the coolingrate that yields maximum viability (i.e. the top of the inverse U)ranging over many orders of magnitude 1-1000° C./minute.

Cellular injury mechanisms may depend on the thermal history that a cellexperiences during freezing. This thermal history is defined by fourthermal parameters: cooling rate (CR), end (or minimum) temperature(ET), time held at the minimum temperature (hold time, HT), and thawrate (TR), all of which have been linked to injury. It has been found inAT-1 tumor cells that ET and HT are the most injurious in cellsuspensions. However, each cell type investigated typically has a uniquethermal threshold where AT-1 cells can survive to −80° C. and ELT-3uterine fibroid cells can survive only to −30° C. with other thermalparameters being similar, and, as indicated above, if the rates ofcooling and heating (CR, TR) are sufficiently high, cell damageirrespective of the ET and HT may be obtained.

Treating biological material by contacting the material with thecompositions of the invention, thereby inducing inflammation in thelocalized region of the material, is also believed to cause variouschanges in the biological material. For example, it is believed thatsuch treatment may be effective in changing a destruction point of thebiological material in a localized region of a mammal. As used herein,the “destruction point” is understood to mean the temperature at whichthe biological functions of the biological material undergoing thethermal surgical procedure are rendered irreversibly inoperative, eitherduring the procedure or shortly thereafter, preferably within three daysor less, more preferably within two days or less, even more preferablywithin one day or less, and still more preferably within 12 hours orless of the procedure. In some instances, the functions of thebiological material may be rendered irreversibly inoperative within 2hours or less of the procedure, and preferably within one hour or lessof the procedure. In other words, the destruction point is thetemperature, the hold time at a given temperature, and/or the rates ofheating and/or cooling at which cell death results in the localizedregion of the biological material. These temperatures can include thosethat are below normal physiological temperatures and those that areabove normal physiological temperatures, and the destruction point isvariable, depending upon the biological materials of interest.

Without being held to a particular theory, it is believed that sotreating the biological material induces more effective killing at theedge of the treated area, such as through endothelial injury andmicrovascular shut-down post freeze at the edge of the ice ball in acryosurgical procedure. One may be able to visualize the edge of the iceball using monitoring techniques such as NMR, CT, or ultrasound.Therefore, by using an inflammatory agent to provide destruction ofbiological material out to the edge of the ice ball, it may be possibleto visualize the region of injury intraoperatively rather thanpost-operatively in follow-up, thus potentially increasing the controland effectiveness of the thermal surgical technique.

The inflammatory inducing compounds of the present invention areconsidered herein to be adjuvants that enhance the thermal surgicalprocedure. As used herein, to “enhance a thermal surgical procedure” isconsidered to include, but not be limited to, increasing a percentage ofcell death in the localized region of the biological material within agiven time period as compared to untreated regions of the biologicalmaterial.

Alternatively, a thermal surgical procedure may be enhanced byattempting to control the injury. This may be accomplished in acryosurgical procedure, for example, through the use of any of a classof compounds used to diminish the injury, rather than to augment it.These compounds are cryoprotective agents and include, for example,glycerol, dimethylsulfoxide, various sugars, various alcohols, andvarious polymers such as PVP and HES, may enhance the surgical procedureby, in essence, “sculpting” the ice ball from the outside rather thancontrolling it from within the ice ball.

The compositions of the present invention include at least one compound,and may include more than one of any of a number of compounds capable ofinducing some degree of inflammation in at least a portion of thetreated (i.e., contacted) biological material of a mammal, wherein thematerial is identified to undergo a thermal surgical procedure. Forexample, the compounds may include, but are not limited to, one or moreviruses, one or more bacteria, ethanol, cytokines such as TissueNecrosis Factor-alpha (TNF-alpha) or truncated versions of TNF-alpha,bacterial lipopolysaccarides (LPS), interleukins such as IL-1 beta andIL-8, chemokines which recruit white blood cells, oxygen-free radicals,and combinations thereof.

Selection of the one or more inflammatory inducing compounds may takeinto consideration the individual effects and traits of the compound.TNF-alpha, for example, is known to promote inflammation, endothelialinjury, and apoptosis, and may be used alone or in combination withother compounds to provide the desired benefit. TNF-alpha is produced bya number of different cell types, macrophages, tumor and stromal cellsand is thought to be responsible for manifestation of autoimmune andchronic inflammatory disorders. As discussed hereinbelow, in oneembodiment of the invention, TNF-alpha may be directly injected into thebiological material of interest (e.g. a tumor) in order to increase theefficacy of the thermal surgical procedure. Alternatively, in a furtherembodiment, cells that produce TNF-alpha might be directed to, orinjected into, the biological material of interest to increase theefficacy of the thermal surgical procedure through inflammation of thebiological material.

There are various methods for delivering the composition to thebiological material of interest, such methods being an issue ofselecting an appropriate drug delivery system. One method includes theaddition of one or more carriers, particularly carriers that may havespecific receptors for the tumor or tissue of interest. Thus, theinflammation inducing compositions of the present invention mayoptionally include a pharmaceutically acceptable carrier for delivery tothe material of interest, which carrier may also optionally havespecific receptors for the tumor or tissue of interest.

As used herein, a pharmaceutically acceptable carrier may include, butis not limited to, liquid solvents in which the inflammation inducingcompound can be at least partially suspended and/or diluted, such as asaline solution, and any other carrier which may provide for directinterstitial injection in liquid suspension, IV or IP injection,impregnation of the composition into microbeads or nanobeads to beinjected locally or systemically and then targeted, gelfoam, retroviralDNA injections (gene therapy), etc., and combinations thereof.

Pharmaceutically acceptable carriers may also optionally includebuffering agents, as are known, to ensure the resulting inflammationinducing composition has a pH value within a range acceptable forphysiological use. Such agents may include, but are not limited tophosphate buffered solutions.

The inflammation inducing compositions of the present invention may alsoinclude further components to provide additional benefits. For example,additional components may include, but are not limited to, a compositionto further enhance cell and tissue destruction by cryosurgery. U.S. Pat.No. 5,654,279 to Rubinsky et al. provides one example of possibleadditional additives. An additional example includes additives that mayprovide for eutectic freezing in the biological material 12 as providedin U.S. Ser. No. 10/461,763 entitled CRYOSURGERY COMPOSITIONS ANDMETHODS, filed on Jun. 13, 2003 (Atty. Docket 110.01920101). Inaddition, chemotherapeutic agents can also be introduced with theinflammation inducing composition.

As discussed in more detail below, the location and/or extent to whichthe inflammation inducing composition may be infused into the tissue canbe monitored through any number of known techniques. The inflammationinducing compositions may, therefore, optionally include compounds toassist in visualization and monitoring. For example, compounds and/orsolutions that may enhance ultrasonic imaging, fluoroscope, MRI,impedance technique (e.g., U.S. Pat. No. 4,252,130 to Le Pivert), etc.,can be added to the inflammation inducing composition to allow forvisualization of the location of the inflammation inducing composition.Examples include, but are not limited to, contrast agent added with salt(i.e., hypaque) and/or the inflammation inducing compounds, salt and/orthe inflammation inducing compounds tagged with a fluorescent marker,ultrasound contrast agents, and use of an impedance metric device to seehow impedance changes locally with infusion.

The time interval for treating the biological material with thecomposition of the present invention prior to performing a thermalsurgical procedure can range from, e.g., a matter of minutes, hours, ordays, depending on the composition and biological material of interest,and the required time interval is measured according to theeffectiveness of the kill. However, it is currently believed thatimproved effects may be provided if at least about one hour or moreelapses between delivery of the composition to the biological materialand the thermal surgical procedure. As an example, it may be possible touse a four (4) hour time interval for treating biological material withthe composition of the present invention prior to performing the thermalsurgical procedure. This time may, however, change depending upon anynumber of factors, including but not limited to, the type and locationof the biological material, the inflammatory composition used (and/orits delivery system), and the existing physiological state of thebiological material.

Without being held to any particular theory, it is believed that theinflammatory response typically should be sufficiently activated withinthe endothelium of the microvasculature to give the augmented injuryresponse after cryosurgery or thermosurgery. The inflammatory responseas measured by adhesion molecule production within endothelium (VCAM andICAM) may typically take several hours to peak. However, in certaininstances it may be true that much shorter times, such as on the orderof minutes, will provide the desired response.

The methods of the present invention include methods of performing athermal surgical procedure in which the biological material identifiedto undergo the procedure is contacted with an inflammation inducingcomposition of the present invention, and the temperature of thebiological material is adjusted. The temperature is adjusted such thatthe material is either cooled to below or heated to above aphysiological temperature to destroy at least a portion of the material.The composition selected is a type such that, for a specified time andamount of the composition, inflammation is induced in at least a portionof the biological material, and such inflammation is induced. Suchtreatment of the material (i.e., contact with the composition) may occureither during the surgical procedure, before the procedure, after theprocedure, or any combination thereof.

Treatment of the biological material is considered to include, but notbe limited to, one or a combination of means for delivering thecomposition to at least a portion of the identified biological material.Such delivery means may include, but not be limited to, introduction ofthe composition into one or more locations of the biological materialthrough the use of hypodermic needles, introduction via of one or moreneedles integrated into or attached to a cryoprobe, introduction viadiffusion, and introduction via iontophoresis (or any other use ofelectric fields to drive solution flow in tissues), direct interstitialinjection in liquid suspension, IV or IP injection, impregnation of thecomposition into microbeads or nanobeads to be delivered locally orsystemically and then targeted, retroviral DNA injections (genetherapy), etc., and combinations thereof.

Alternatively, the delivery means could involve incorporation of thecomposition into a gel or foam for topical use, or incorporated into animplantable material to be used before or after the thermal surgicalprocedure, for example, a gelfoam, a tissue engineered collagen, fibrinbased product, etc.

Biological materials to be treated according to the methods and systemsof the present invention typically have a destruction point, that is, atemperature, or the hold time at a given temperature, and/or the ratesof heating and/or cooling at which cell death results in the localizedregion of the biological material, with the temperature typically aboveor below physiological temperatures, that is, temperatures at which thebiological functions of the biological material undergoing the thermalsurgical procedure are rendered irreversibly inoperative (i.e., celldeath in the localized region of the biological material). Without beingheld to any particular theory, it is believed that the present inventionoperates, at least in part, to change the destruction point of, forexample, a localized region of a mammal that includes biologicalmaterials identified to undergo a thermal surgical procedure.

The present invention is believed to provide improved assessment of theactual location of cell and/or tissue death in identified biologicalmaterials. Such assessment is aided through the use of known monitoringtechniques that may locate and/or determine the extent to which theinflammation inducing composition has been infused into the tissueincluding, for example, ultrasonic imaging, fluoroscope, MRI, andimpedance techniques, particularly when used in conjunction with acomposition including an imaging enhancing compound such as vascularperfusion contrast agents. Thus, by being able to visualize, forexample, the edge of an ice ball during a cryosurgical procedure, theedge of the injury during the procedure may be monitored during theprocedure, providing improved intraoperative imaging and injuryassessment.

The present invention further provides systems for inducing inflammationin at least a portion of biological material intended to undergo athermal surgical procedure that includes a composition of the presentinvention and means for delivering the omposition to the biologicalmaterial. Such delivery means may include, for example, delivery of thecomposition through a catheter (e.g., through a lumen, in a balloon orother chamber positioned at a desired location, etc.), delivery via aneedle, and delivery via a thermal surgical probe adapted to transferthermal energy, such as, e.g., a cryoprobe.

The composition may be delivered either directly to the site of thethermal surgical procedure, or may be delivered to another location,such as, e.g., a site adjacent to the location of the thermal surgicalprocedure, or may be delivered systemically when appropriate. Further,the inflammation inducing composition may be delivered to the biologicalmaterial before, during, and/or after a thermal surgical procedure.

Systems of the present invention may further include the use of athermal energy transfer means that provides thermal energy to biologicalmaterial (a heat source) or that removes thermal energy from biologicalmaterial (a heat sink). Such means may include, for example, thermalsurgical probes, catheters, implantable devices, etc. An effective meansof providing and/or removing thermal energy in the methods and systemsof the present invention may be a thermal surgical probe, wherein theprobe is effective for either removing thermal energy from or supplyingthermal energy to, depending on the type of thermal surgical procedurecontemplated, at least a portion of the biological material of interestat a rate to provide heating or cooling, resulting in at least partialdestruction of biological material at the location of the thermalsurgical procedure. Such probes may include, for example, catheters,hollow needles, cryoprobes, implantable devices, etc.

The present invention may include a kit for use in a thermal surgicalprocedure. The kit may include a thermal surgical probe that is adaptedto transfer thermal energy as appropriate, either by removing thermalenergy for use in a cryosurgical procedure, or by supplying thermalenergy, for use in a thermosurgical procedure. The kit preferablyincludes a composition that includes at least one compound effective forinducing an inflammatory response in biological material identified toundergo a thermal surgical procedure. Such compositions may include oneor more of compounds that are selected from the group of at least onevirus, at least one bacterium, ethanol, cytokines, interleukins,chemokines, oxygen-free radicals, bacterial lipopolysaccharides, and anycombination thereof. If a cytokine is selected for use, it may bepreferred that the cytokine used is TNF-alpha, truncated versions ofTNF-alpha, and any combination thereof. If an interleukin is selectedfor use, it may be preferred that the interleukin used is IL-beta, IL-8,and any combination thereof. The composition may further include anoptional pharmaceutically acceptable carrier and/or any of the optionalconstituents previously discussed. The probe that is used is any thatmay be adapted for use in a thermal surgical procedure such as, but notlimited to, a catheter, a hollow needle, a cryoprobe, an implantablemedical device, etc.

As an example, FIG. 1 shows one possible embodiment of a system 10according to the present invention for inducing inflammation inbiological material 12 that includes a portion 14 that has beenidentified to undergo a thermal surgical procedure. As discussed herein,the biological material 12 can include a tissue, including cells,intended to undergo, at least in part, a thermal surgical procedure. Theportion 14 of biological material 12 can have a similar cell and/ortissue structure as the surrounding segment of biological material 12.Alternatively, the portion 14 can have one or more morphologicallydistinct cell and/or tissue structures as compared to the remainingsegment of the biological material 12. In one example, the portion 14can be a tumor.

The inflammatory state of the portion 14 of the biological material 12can be changed relative to the remaining segment of the biologicalmaterial 12 through the use of the inflammatory inducing composition ofthe system 10 of the present invention. The biological material 12 maybe treated with the inflammation inducing composition of the presentinvention for a time, in an amount and of a type effective to induceinflammation in at least a portion of the identified biologicalmaterial. In one example, the inflammation inducing composition can beone or more of the compounds for inducing an inflammatory response asdiscussed herein.

The portion 14 of the biological material 12 to be treated with theinflammation inducing composition as a part of the thermal surgicalprocedure may be identified by any number of known techniques. Forexample, tumor structures may be identified through tissue structure,biological markers, ultrasound, or any number of other techniques.Furthermore, the location and/or extent to which the inflammationinducing composition has been infused into the tissue (e.g., the portion14 in FIG. 1) can also be monitored through any number of techniques,and one or more identification and/or monitoring techniques may be usedas required.

Once identified, the inflammation inducing composition can be deliveredto the portion 14 of the biological material 12. In one possibleembodiment, the composition can be delivered through the use of deliverydevice, such as, e.g., a catheter 16. In general, the catheter 16includes a lumen, where the inflammation inducing composition can movethrough the lumen of the catheter 16 and into the biological material 12in which the inflammatory response is desired. The catheter 16 of thepresent invention may also include a needle at a distal end of thecatheter 16 for delivering the inflammation inducing composition.Alternatively, the catheter 16 can further include a trocar in the lumenof the catheter 16 to facilitate delivering a portion of the catheter 16to the biological material 12 in which the inflammatory response isdesired. U.S. Pat. No. 5,807,395 provides some examples of catheters 16that may be suitable for injecting the inflammation inducing compositionof the present invention.

The system 10 may also include one or more probes 18, where the probes18 can remove and/or deliver thermal energy from the location forthermal surgical procedure at a rate sufficient to cause biologicalmaterial 12 at the location for thermal surgical procedure to undergocooling or heating. In one embodiment, heat may be removed at a ratesufficient to cause cooling of the tissue surrounding the probe at a1-100° C. per minute rate. In an additional embodiment, thermal energymay be supplied at a rate sufficient to cause heating of the tissuesurrounding the probe at a 1-100° C. per minute rate. In someembodiments, the catheter 16 (or other device) used to deliver theinflammation inducing composition may also be used to deliver or removethermal energy, as discussed herein.

Other rates are also contemplated in the methods and systems of thepresent invention, depending on the circumstances of use. During acryosurgical procedure, for example, intracellular ice formation mayoccur at higher rates, typically greater than about 30° C./minute, moretypically greater than about 50° C./minute. This mechanism of injury mayoccur proximate the cryosurgical probe; however, as rates typicallydecrease quickly moving away from the probe, the effects of a broadrange of rates may be more strongly felt proximate the probe, while therates in the outer areas of the biological material being treated may belower (e.g., in the range of 1-10° C./minute) regardless of the rate atwhich the probe, or other device, is cooled or heated.

One or more probes 18 may be used to cool and/or heat the biologicalmaterial 12 at a rate effective to destroy at least the portion 14 ofthe treated biological material. In a further embodiment, for example,when the biological material 12 is cooled with the probe 18, an ice ballis formed. The ice ball formation typically originates proximate the tipof each probe 18. As thermal energy is removed from the tissue, the iceball grows. Visualizing the size of the ice ball formation may assist indetermining the extent, or amount, of tissue and cell material killedduring a thermal surgical procedure. Visualization of the size of theice ball may be accomplished, e.g., through the use of hypaque withfluoroscopy, ultrasonic imaging, MRI, gadolinium with MRI, impedancetechniques, or other applicable techniques.

FIG. 2 depicts one example of the relationship of temperature versusdistance from the ice ball center. Line 100 illustrates the distancefrom the center of the ice ball (e.g., the location of the probe) wherecell death will typically occur for biological material that has notbeen treated with the inflammation inducing composition. As will benoted, the temperature at the distance where the cell death is suggestedto occur within tumors is between approximately −20° C. to approximately−60° C. in the depicted example. In contrast, when the biologicalmaterial is treated with the inflammation inducing composition asdescribed herein, the distance from the center of the ice ball (e.g.,the location of the probe) where cell death will typically occur may beincreased along with the temperature at which this cell death occurs.This is illustrated by line 120. Thus, the inflammation inducingcomposition may effectively increase the distance from the probe, orother device, for which cell death will typically occur without acorresponding increase in the diameter of the ice ball.

In addition to increasing the volume in which cell death will typicallyoccur, the use of the inflammation inducing composition is believed toenable a change in the size or extent of the ice ball such that the useof the composition may, for example, reduce the size of the ice ball.Although not wishing to be bound by any particular theory, it isbelieved that this may be due, at least in part, to the preconditionedstate of the microvasculature of the biological material caused by theintroduction of the inflammation inducing composition. A reduction inthe size of the ice ball formation coupled with the increase in thevolume within which cell death will typically occur in the cryosurgicalice ball results in an ice ball with a size that more closely correlatesto the volume in which the actual cell death occurs. Further, theinflammation composition is believed also to enable a change of thetemperature at which cell death occurs (the “destruction point”) byaugmenting the injury zone such that it more closely matches the iceball or, alternatively, by altering the phase change temperature,thereby potentially decreasing the ice ball size for a given probeoperation.

FIG. 3 illustrates what is believed to occur with respect to the size ofan ice ball according to the methods, compositions, and systems of thepresent invention. The ice ball is shown generally at 150. Probe 160 isused to remove thermal energy from the biological material so as tocreate the ice ball 150. When the ice ball is formed in biologicalmaterial not treated with the inflammation inducing composition, a killzone 170 surrounds the tip of the probe 160, within a boundary 180 ofthe ice ball 150. As FIG. 3 illustrates the boundary 180 of the ice ball150 is located at a distance 190 from the edge of the kill zone 170.

In contrast, when the biological material is treated with theinflammation inducing composition according to the present invention,the kill zone 200 may thereby be enlarged as compared to kill zone 170.In addition, the boundary 210 of the ice ball 150 may be reduced ascompared to the boundary 180. Thus the size of the ice ball may bereduced and the size of the kill zone within the ice ball may beincreased due to administration of the inflammation inducing compositionof the present invention. One potential beneficial result of thesechanges in kill zone and ice ball size is that the kill zone may moreclosely correlate with the size of the ice ball. This may allow surgeonsto more closely predict the actual kill zone created during the thermalsurgical procedure and more effectively treat diseased tissues whilepreserving adjoining normal tissues from injury.

For example, during cryosurgery of the prostate and many other organssuch as liver, kidney or brain, ultrasound or MRI can be used to monitorthe extent of the cryosurgical ice ball and it is used at some level topredict the outcome of the procedure. The ice ball boundary, however, istypically at a temperature of approximately −0.5° C., while thresholdsof prostate cancer destruction are reported anywhere from approximately−20° C. to −60° and in some instances even lower (Hoffmann N, Bischof J.2002. Urology 60 (Supplement 2A): 40-9; Saliken J, Donnelly B, RewcastleJ C. 2002. Urology (Supplement 2A): 26-33). Thus, while monitoring isuseful for imaging the ice ball and predicting likely outcome of thesurgery, it may not assist in the outcome that not all of the tissuethat is frozen is also effectively treated. In some tissues, such asliver and sometimes kidney, the ice ball may be allowed to progress intoa margin of normal tissue beyond the tumor. However, this is not thecase with prostate since overfreezing into sensitive adjacent structuressuch as the rectum and urethra can cause complications such as rectaland urethral fistulas. On the other hand if the surgeon is tooconservative and under freezes by keeping the ice ball solely within theprostate, then cancer which often exists under the prostate capsule atthe edge of the gland may not be effectively treated leading in somecases to recurrence of disease. One approach of the present inventionmay include the use of cryosurgical adjuvants in the form of both aninflammation inducing composition and a eutectic freezing point changingagent (such as, e.g., those described in U.S. patent application Ser.No. 10/461,763, entitled CRYOSURGERY COMPOSITIONS AND METHODS, filedJun. 13, 2003 (Attorney Docket No. 110.01920101) may be used together.The combination may provide inflammation to the biological material toboth increase freeze destruction (salts and TNF-alpha) and reduce thetemperature at the edge of the ice ball. The combination may improve theeffectiveness and predictability of the kill zone while preservingnormal tissues from excessive and/or unnecessary injury.

A composition including the cytokine TNF-alpha was used to increase thethreshold temperature of destruction after cryosurgery in human prostatecancer (LNCaP grown in nude mice) to a mean temperature above 0° C. Thelocal use of TNF-alpha to pre-inflame prostate cancer increased theability of freezing to destroy the cancer. Thus, monitoring techniquessuch as ultrasound, CT, MR, and others which focus on the edge of theice ball may, in the presence of TNF-alpha, also be capable ofpredicting the outcome of the treatment by measuring the edge of theinjury at the same time that the edge of the ice ball is measured.

Pre-inflammation of biological tissue may be a mechanism useful inaccentuating vascular injury during thermal surgical procedures. Inaddition, there may be a role for the endothelium in shutting down themicrovascular supply to prostate cancers (Dunning AT-1) grown inCopenhagen rats fitted with dorsal skin fold chambers (DSFC) (HoffmannN, Bischof J. 2002. Urology 60 (Supplement 2A): 40-9). Data from thesestudies indicates that the tumor could under some freeze/thaw conditionssurvive freezing to −80° C. and below in vitro, but that moderatefreezing and thawing to about −20° C. leads to vascular stasis andhistological necrosis by ischemia as assessed at day 3 after the freezein both the cancer and normal rat skin in vivo.

The accentuation of the vascular mechanism of injury has been approachedby focusing on inflammation. The role of the endothelium suggested thepossibility of creating a pre-existing non-destructive inflammationwithin the tissue prior to the freeze. The cytokine TNF-alpha is knownto upregulate NF-kB and various adhesion molecules within endotheliumand has also recently been used in the DSFC (Fukumura D et al. 1995.Cancer Research 55: 4824-9). As discussed herein, local TNF-alphadelivery is an effective way to achieve pre-inflammation prior to athermal surgical procedure.

EXAMPLES

The following are examples are provided to illustrate the presentinvention and are not intended to limit the present invention thereto inany manner

Example 1

The dorsal skin flap chamber (DSFC) of male athymic nude mice was seededwith the tumor LNCaP Pro 5 human prostate cancer, inflamed withTNF-alpha, subjected to cryosurgery, and assessed according to thefollowing procedure.

The dorsal skin of a male athymic nude mouse was sandwiched between twoidentical anodized aluminum frames, a 19 millimeter (mm) by 22 mmchamber was mounted onto the mouse by three screws, the skin wasattached to the chamber with 4-O silk using suture holes, and the skinon the side of the viewing region was removed, exposing the dermiscontaining the microvasculature on the opposite side of the skin.

To provide chambers having tumors, approximately 5×10⁶ LNCaP Pro 5 humanprostate tumor cells were mixed with MATRIGEL matrix (BD Biosciences,Bedford, Mass.) as described by Lim et al. (Prostate, 22:109-118(1993)). Approximately 30 μl of the cell suspension was applied to thesurface of the microvascular bed immediately after the initial chamberimplantation and the tumor was allowed to grow for 10 days, the skinreaching a total thickness of about 450 micrometers (μm) and the tumorextending approximately 10 mm in diameter.

A local application of 20 μl of a 10 ng/ml TNF-alpha (total applicationof 0.2 ng) was applied to the tumor tissue within the DSFC for about 15minutes, after which the TNF-alpha solution was wicked off and thetissue was covered with a glass window. After four (4) hours, the mousewas anesthetized, TNF-alpha-induced inflammation was measured byobservation of leukocyte rolling, and thereafter cryosurgery wasimmediately performed in the inflamed tissue.

The cryosurgery was performed with an argon-cooled, 5 mm diametercryoprobe (EndoCare, Irvine, Calif.) activated for a cooling time of 5minutes and a target temperature of −160° C., which corresponded to anaverage external probe end temperature of about −125° C. Type “T”thermocouples (Omega Tech. Corp, Stamford, Conn.), having a 0.5 mm beaddiameter, were inserted into the tissue and were used to measure theaverage external probe end temperature. The temperature at eachthermocouple was recorded using a HYDRA DATA LOGGER SERIES 2 (Fluke,Everett, Wash.).

After the cryosurgery the probe was turned off and the tissue wasallowed to thaw passively at room temperature.

The vasculature was imaged using a 70 kD fluorescein isothiocyanate(HITC)-labeled dextran (Molecular Probes, Eugene Oreg.). At 3 dayspost-treatment, 0.05 ml of a solution of FITC-labeled dextran (10 mgdextran/mi PBS (Gibco BRL, Gaithersburg, Md.)) was injected into thetail vein of the mouse. The dorsal skin flap chamber was thenilluminated with a mercury lamp and a FITC signal-enhancing filter(λ=470-490 nm) to view the contrast fluorescence. A Silicon IntensifiedTransmission camera (Hamamatsu, North Central Instruments, Twin Cities,Minn.) was used to detect the fluorescent signal, and the signal wasrecorded with a JVC S-VHS video recorder (JVC Company of America,Aurora, Ill.).

Hstological analysis of the entire tissue was performed at day 3post-cryosurgery according to Hoffmann et al. (ASME J. BiomechanicalEngineering 123:310-316 (2001)), and images of the histology were takenon an Olympus BX-50 upright microscope (Leeds Precision Instruments,Minneapolis, Minn.). The end temperature of the cryolesion and thethermal parameters within the cryosurgical ice ball were calculatedaccording to the methods of Chao et al. (Cryobiology, 2004 (in press)).

The above example was repeated a total of 9 times to provide data forTNF-alpha-treated tumor tissue, and was repeated a total of 13 timeswithout TNF-alpha treatment.

Results:

The area of vascular injury was observed with FITC-labeled dextran. Theresults showed a substantially complete destruction of the vasculaturein the center of the lesion and an abrupt change to normal patencymoving radially outward. It was determined that regions of vascularstasis lead directly to tissue necrosis. Further, the edge of the staticzone (i.e., the zone of vascular stasis) at day 3 post cryosurgery intissues inflamed with TNF-alpha was at a radius greater than that fortissues that were not inflamed with TNF-alpha (r=3.81±0.29 mm in LNCaPPro 5 tumor tissues without TNF-alpha treatment and r=4.07±0.34 mm intissues with TNF-alpha treatment). Additionally, the edge of the staticzone extended beyond the edge of the ice ball for LNCaP Pro 5 tumortissues that were treated with TNF-alpha, whereas the edge of the staticzone stayed within the edge of the ice ball in inflamed normal skintissues that were treated with TNF-alpha (Example 2, below).

The minimum temperature required for causing necrosis was 3.5±6.9° C. inTNF-alpha-treated LNCaP Pro 5 tumor tissue. Compared to tissues withoutTNF-alpha treatment, where the minimum temperature required for causingnecrosis was −16.5±4.3° C. in LNCaP Pro 5 tumor tissue, the resultsindicate that the local use of TNF-alpha can increase the thresholdtemperature of cryo-destruction by more than 10° C.

Example 2

The procedure according to Example 1 was repeated, with the exceptionthat the biological material treated was normal nude mouse hypodermis(i.e., normal skin).

Example 2 was repeated a total of 9 times to provide data forTNF-alpha-treated normal tissue, and was repeated a total of 14 timeswithout TNF-alpha treatment.

Results:

Similar, results were obtained in normal nude mouse hypodermis withouttumors. Inflammation induced by TNF-alpha moved the edge of the staticzone closer to the edge of the cryosurgical ice ball (r=3.99±0.13 mm intissues treated with TNF-alpha, as compared with r=3.13±0.39 mm intissues without TNF-alpha treatment), and the minimum temperaturerequired for cryo-destruction was −9.8±5.8° C. in TNF-alpha-treatednormal skin as compared with −24.4±7.0° C. in untreated normal skin.

Cell suspensions of human endothelial cells (MVECs) were used to assessthe enhancement of direct cellular injury (DCI) by use of an adjuvant,wherein the adjuvant used is TNF-alpha.

Example 3

Measuring Enhancement of DCI in MVEC Endothelial, MCF-7 Breast Cancerand LNCaP Pro 5 Prostate Cancer cells by TNF-alpha addition.

Human dermal microvascular endothelial cells (MVEC) were prepared andgrown as adherent monolayers as described by Gupta et al., ExperimentalCell Research, 230:244-251 (1997), maintained in a 37° C./5% CO₂/95%humidified air environment in T-flasks pre-coated with 1% gelatin inMCDB 131 medium supplemented with 20% heat-inactivated human male serum,hydrocortisone, cAMP, L-glutamine, heparin, endothelial cell growthsupplement (Vec Tec, Schenectady, N.Y.) and antibiotics. LNCaP cellswere cultured as adherent monolayers in DMEM/F12 medium supplementedwith 10% FBS, antibiotics and dihydrotesterone (DHT). MCF-7 cells weregrown in similar medium with the following exception: 5% (rather than10%) FBS, antibiotics and insulin (no DHT). Cells were subcultured byrinsing with Hank's balanced salt solution (HBSS), followed by lighttrypsinization, enzyme neutralization, and reseeding.

One to three days prior to TNF-alpha exposure and/or freezing, cellswere reseeded onto 96-well plates for apoptosis/necrosis assay and ontoT-flasks for EMSA or Western blots, as monolayers or in Petri dishes ascollagen gels (live-dead assay). Each sample was then exposed to mediumwith TNF-alpha at concentrations from 10 nanogram per milliliter (ng/ml)to 1 microgram per milliliter (μg/ml) for 4 to 48 hours.

Control and experimental FIT groups were then assessed for cellviability at varying time points starting 3 hours after intervention.Cell viability was measured microscopically by a fluorescent dye assay(cell viability assay) by an apoptotic/necrotic assay.

The fluorescent dye assay was used to assess the plasma membraneintegrity of cells immediately before (control) and after F/T usingHoechst 33342 and propidium iodide (PI). Each dye has affinity tonucleic acid, i.e. all cells regardless of viability take up Hoechst andonly plasma membrane compromised cells take up PI. Cells were incubatedwith 9 μM Hoechst 33342 and 7 μM PI for 15 minutes at 37° C., placed ona microslide, cover-slipped and the percentage of dead cells/fielddetermined at 200× using a fluorescent microscope (Olympus BX-50, Tokyo,Japan).

The apoptosis/necrosis assay was used to map thermal/adjuvant conditionsthat triggered apoptosis. The cells were stained with fluorescentAnnexin V.

The fluorescent dye assay was performed with at least 5 representativefields and a total of 100-200 cells/sample were counted. All sampleswere measured in four or six replicates and the resulting values wereaveraged.

Results:

TNF-alpha was shown to increase cryosensitivity of the MVEC, MCF-7 andLNCaP Pro 5 cells in vitro. The action of the TNF-alpha predominantlyinflamed cells, and the inflamed cells exhibited increased necrosis invitro.

All references identified herein are incorporated by reference in theirentirety as if each were incorporated separately. This invention hasbeen described with reference to illustrative embodiments and is notmeant to be construed in a limiting sense. Various modifications of theillustrative embodiments, as well as additional embodiments of theinvention, will be apparent to persons skilled in the art upon referenceto this description.

1-6. (canceled)
 7. A composition comprising at least one compoundeffective for inducing an inflammatory response in biological materialidentified to undergo a thermal surgical procedure.
 8. The compositionof claim 7 wherein the at least one compound effective for inducing aninflammatory response is selected from the group consisting of at leastone virus, at least one bacterium, ethanol, cytokines, interleukins,chemokines, oxygen-free radicals, bacterial lipopolysaccharides, andcombinations thereof.
 9. The composition of claim 8 wherein the cytokineis selected from the group consisting of TNF-alpha, truncated versionsof TNF-alpha, and combinations thereof.
 10. The composition of claim 8wherein the interleukin is selected from the group consisting ofIL-beta, IL-8, and combinations thereof.
 11. The composition of claim 7further comprising a pharmaceutically acceptable carrier.
 12. Thecomposition of claim 11, wherein the pharmaceutically acceptable carrieris selected from the group consisting of a saline solution,encapsulation in microbeads, encapsulation in nanobeads, retroviral genetherapy, impregnated gelfoam, and combinations thereof.
 13. Thecomposition of claim 7 further comprising a compound selected from thegroup consisting of a buffering agent, a chemotherapeutic agent, a salt,a contrast agent, a fluorescent marker, an impedance metric device,ultrasound contrast agents, and combinations thereof. 14-31. (canceled)